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PRODUCTION ANIMALS
PRODUCTION ANIMALS
Pharmacokinetics of bromide in adult sheep following oral and intravenous administration TA Quast, MD Combs and SH Edwards*
Objective To determine the pharmacokinetics of bromide in sheep after single intravenous (IV) and oral (PO) doses. Procedure Sixteen Merino sheep were randomly assigned to two treatment groups and given 120 mg/kg bromide, as sodium bromide IV or potassium bromide PO. Serum bromide concentrations were determined by colorimetric spectrophotometry. Results After IV administration the maximum concentration (Cmax) was 822.11 ± 93.61 mg/L, volume of distribution (Vd) was 0.286 ± 0.031 L/kg and the clearance (Cl) was 0.836 ± 0.255 mL/h/ kg. After PO administration the Cmax was 453.86 ± 43.37 mg/L and the time of maximum concentration (Tmax) was 108 ± 125 h. The terminal half-life (t½) of bromide after IV and PO administration was 387.93 ± 115.35 h and 346.72 ± 94.05 h, respectively. The oral bioavailability (F) of bromide was 92%. No adverse reactions were noted in either treatment group during this study. The concentration versus time profiles exhibited secondary peaks, suggestive of gastrointestinal cyclic redistribution of the drug. Conclusions and clinical relevance When administered PO, bromide in sheep has a long half-life (t½) of approximately 14 days, with good bioavailability. Potassium bromide is a readily available, affordable salt with a long history of medical use as an anxiolytic, sedative and antiseizure therapy in other species. There are a number of husbandry activities and flock level neurological conditions, including perennial ryegrass toxicosis, in which bromide may have therapeutic or prophylactic application. Keywords
bromide; pharmacokinetics; sheep
Abbreviations AUC0-∞, area under the curve; AUMC0-∞, area under the first moment curve; Cmax, maximum concentration; λz, terminal elimination rate constant; ECF, extracellular fluid; LD, loading dose; PK, pharmacokinetics; PD, pharmacodynamics; PRGT, perennial ryegrass toxicosis; t½, terminal elimination half-life; Tmax, time to maximum concentration; Vd, initial volume of distribution; Vz, terminal volume of distribution. Aust Vet J 2015;93:20–25
doi: 10.1111/avj.12285
B
romide has been a therapeutic option for human epilepsy since its efficacy in the management of seizures was first discovered in the middle of the 19th century.1 In humans it has also been used for a wide variety of other applications, including as a sedative and an anxiolytic.2
*Corresponding author. School of Animal and Veterinary Science, Charles Sturt University, Wagga Wagga, New South Wales, Australia; [email protected]
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In veterinary medicine, potassium bromide (KBr) has found renewed favour in recent years as a therapeutic agent. In Australia, KBr is registered in dogs for the treatment of idiopathic epilepsy and it is being increasingly used worldwide for seizure therapy as it is associated with fewer side-effects than phenobarbitone.3–6 It is also used extensively off-label by horse owners as a calmative for nervous horses; however, to date little research has been undertaken as to its efficacy and appropriate application.7,8 The potential uses of Br in sheep have not been previously investigated. Because of its tranquillising and anxiolytic actions, Br has the potential to address animal welfare and production issues associated with a range of husbandry procedures such as road and sea transportation, anxiety associated with shearing and inanition associated with stress and introduction of new feeds.9–11 Bromide also has a potential use as a therapeutic agent for neurological diseases that affect sheep at the herd level. For example, the perennial ryegrass toxin, lolitrem B, was recently identified as a calciumactivated potassium channels antagonist, depolarising the neuronal membrane and thereby increasing neuronal excitability.12–14 Bromide’s action via chloride channels results in membrane hyperpolarisation and Br also appears to potentiate GABAergic inhibition.1,6,15–18 These results suggest Br could be used prophylactically to reduce the incidence of perennial ryegrass toxicosis (PRGT) associated with lolitrem B. The distribution of Br in sheep has been partially elucidated; IV 82Br was used to estimate ovine extracellular fluid (ECF) volume and was found to exhibit delayed distribution kinetics.19,20 Essentially, Br is initially confined within the vascular space, with extravascular movement into interstitial fluid being slow, taking 2–3 h to equilibrate with the ECF. Its equilibration with rumen fluid is even slower, with only 4–5% entering the rumen at 3 h and taking 24 h for full equilibration to be reached. In the course of drug development, both pharmacokinetic (PK) and pharmacodynamic (PD) studies are conducted in order to guide determination of rational dosage schedules (dose and administration interval) for subsequent clinical trials. From PK studies in a small number of animals, using a test dose (not necessarily a predicted therapeutic dose), the concentration versus time profile is generated. From these simple plots, estimates of the necessary PK parameters may be derived, which are subsequently used to determine a dose regimen. Target drug concentrations (within the therapeutic range) are determined from PD studies, involving dose–response experiments (in vitro and in vivo dose titration). The use of such a PK–PD approach offers rational guidance in attempting to streamline the drug development process, with the aim of reducing animal wastage stemming from large-scale dose-titration studies.21–23 Moreover, once the
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Of prime importance in this study was to determine the PK parameters of most clinical relevance. A relatively long terminal elimination half-life (t½) is important for an extended duration of action; t½ is also used to estimate withholding periods, vitally important with any drug used in food animals. Volume of distribution (Vd) is the parameter used to calculate the loading dose (LD), a large dose administered to reach target drug concentrations quickly. For the first time, this study investigated the pharmacokinetics of Br in sheep, as the basis for future studies of its therapeutic applications. Materials and methods Animals Sixteen 6-year-old merino ewes (weight 49.5–67 kg; average body condition score > 2) were allocated equally to IV and PO treatment groups by simple systematic randomisation (all sheep were mixed in a pen then put through a race, with every second sheep assigned to pen B. Allocation of treatment to pen A and B was decided by a coin toss). Animals were placed in individual feeding pens and fed twice daily on a ration of oats and lupins, with ad libitum hay and water. Estimated chloride content of the oats and lupins was 0.11% and 0.4%, respectively. Indwelling 16G, 32-cm IV cannulas (Braun, Certo Splittocan 335, Melsungen, Germany) were placed in the left jugular vein and secured with a 2/0 polypropylene suture. A 25-cm low-volume IV extension set (BMDi TUTA, NSW, Aust) was connected to the catheter hub and the area was then bandaged. Sodium bromide (NaBr; Sigma-Aldrich, St Louis, MO, USA) and potassium bromide (KBr; Sigma-Aldrich) solutions were prepared using sterile water. The prepared NaBr solution was then filtered through a microfilter (0.22 μ, MILLEX GP, Cork, Ireland). Sodium and potassium salts were administered to dose the sheep with 120 mg/kg of Br (154.6 mg/kg NaBr or 178.8 mg/kg KBr). All serum concentrations are for Br. KBr is the most readily available form of Br for oral therapy and NaBr was used for the IV study because of cardiotoxicity associated with potassium. Both salts were fully disassociated in solution, so PK differences were not expected. Administration route Intravenous. The NaBr solution was administered through the cephalic vein using a 21G needle over a period of 1 min. Sheep were restrained in a ‘seated’ position. Blood samples were collected at 0, 1, 5, 10, 15, 20 and 30 min and then at 1, 2, 3, 4, 6, 8, 10, 12 and 24 h. Samples thereafter were collected at 12 h intervals to 240 h and then at 24 h intervals to 336 h. A final sample was taken at 528 h. For each sample, the initial 2 mL of blood collected was discarded and a sterile syringe used to withdraw 5 mL of blood, which was then placed into a plain separator blood tube (Vacuette, Greiner Bio-one, Kremsmünster, Austria). The cannula was flushed with 3 mL of 5% heparinised saline after each collection. Each blood sample was left to stand for 30 min
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before centrifugation at 2000g for 5 min. Serum was harvested and stored at −20°C until analysis. PRODUCTION ANIMALS
PK parameters are known, it is a simple matter to use PK–PD integration to generate a putative dose regimen for multiple applications of a particular drug. Should a therapeutic have a novel application potential, the new target concentration is applied to the existing PKs.
Oral. The KBr solution was administered via an orogastric tube, which was then flushed with 500 mL water. Blood samples were collected at 0, 1, 2, 3, 4, 6, 8, 10, 12 and 24 h, then at 12 h intervals to 240 h and then at 24 h intervals to 336 h. A final sample was taken at 504 h. When collecting blood samples at 1 h through to 10 h the rumen was auscultated over the caudodorsal blind sack to determine if the high salt load had affected rumen motility. Determination of serum bromide concentrations Serum Br concentrations were determined by colorimetric spectrophotometry as previously described,24 with some modification. Briefly, 0.35 mL of serum was added to 3.15 mL of 10% trichloroacetic acid (Sigma-Aldrich) in a 10-mL centrifuge tube, vortexed, then centrifuged for 15 min at 2000g. Next, 2.5 mL of supernatant was mixed with 0.25 mL of 0.5% Au2Cl6 (Sigma-Aldrich) and left to stand for 30 min. Absorbance was measured with a spectrophotometer at 440 nm. The standard curve was linear in the range of 25–5000 μg/ mL, R2 = 0.9992. The lower limit of quantification was 25 μg/mL. Pharmacokinetics Maximum concentration (Cmax) of Br and time to Cmax (Tmax) were determined directly from the data. Other PK parameters were determined for each sheep by non-compartmental analysis using a commercial software program (Topfit 2.0, Gustav Fischer Verlag). Area under the curve (AUC0-∞) and area under the first moment curve (AUMC0-∞) were calculated by the linear trapezoidal rule;25 the terminal elimination rate constant (λz) was calculated by means of loglinear regression. Clearance (Cl) was calculated using the equation:
Cl =
Dose AUC0−∞
Mean residence time (MRT) was calculated using the equation:
MRT =
AUMC0−∞ AUC0−∞
The initial volume of distribution (Vd) was calculated using the equation below, with C0 estimated by extrapolation of the drug disposition curve from 2 h (assumed equilibration with ECF) to time zero.
Vd =
Dose C0
The terminal volume of distribution (Vz) at pseudo-equilibrium was calculated using the equation:
Vz =
Cl λz
The t½ was calculated using the equation:
t1/2 =
0.69315 λz
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PRODUCTION ANIMALS Whereas parameters Cmax, Tmax, and AUC (where bioavailability is not absolute) are expected to differ when given IV or PO, t½ should be the same, regardless of the route of administration. A t-test of the hypothesis of no difference between the t½ population means was performed. All results are expressed as mean ± standard deviation. Results Animals No discernable neurological effects were seen in sheep in the PO group. No alteration in rumen motility was auscultated and animals continued to eat and drink. Assessment of any acute neurological effects correlating with peak Br concentration following IV administration was difficult because the sheep were held in the ‘seated’ position
throughout the initial 20 min for ease of sampling. Following IV injection, all sheep walked back to their individual pens and observers subjectively reported a mild tranquilising effect for approximately 1–2 h post-injection. Pharmacokinetics The relevant non-compartmental pharmacokinetic parameters derived from this study are summarised in Table 1. The concentration– time profiles for IV and PO serum Br are shown in Figures 1 and 2, respectively. The t½ of Br in sheep following PO administration was 14.4 days and the IV t½ was 16.2 days; however, the difference between groups was not statistically significant (T = 0.7832, df = 14, P = 0.4466).
Table 1. Pharmacokinetic parameters (mean ± SD) of bromide after intravenous and oral administration to eight sheep at a dose of 120 mg/kg
Pharmacokinetic variable
Intravenous administration (mean ± SD)
Oral administration (mean ± SD)
822.11 ± 93.61 − 157221.8 ± 52681.53 545.5 ± 226.1 0.836 ± 0.255 0.286 ± 0.031 0.393 ± 0.102 387.93 ± 115.35 −
453.86 ± 43.37 108 ± 124.86 143948.9 ± 26156.16 413.4 ± 150 − − 0.388 ± 0.037 346.72 ± 94.05 0.92
Cmax (mg/L) Tmax (h) AUC0-∞ (mg*h/L) MRT0-∞ (h) Cl (mL/h/kg) Vd (L/kg) Vz (L/kg) t½ (h) F
AUC0-∞, area under the curve; Cl, clearance; Cmax, maximum concentration; F, oral bioavailability; MRT0-∞, mean residence time; t½, terminal elimination half-life; SD, standard deviation; Tmax, time of maximum concentration; Vd, initial volume of distribution, Vz, terminal volume of distribution at pseudo-equilibrium.
Figure 1. Serum concentration (mean ± SD) of bromide after intravenous administration to eight sheep at a dose of 120 mg/kg.
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Figure 2. Serum concentration (mean ± SD) of bromide after oral administration to eight sheep at a dose of 120 mg/kg. Note multiple peaks occurring after 24 h.
There were numerous pronounced peaks in the PO concentration versus time curve following the initial absorption phase. These peaks approached the Cmax seen on day 1, but occurred many days later (Figure 2). Indeed, some sheep had Tmax values well beyond the initial PO absorption phase. Similar but smaller peaks were also seen with IV Br (Figure 1). Discussion Bromide was absorbed rapidly following PO administration. Prior to this study, it was predicted that absorption might be delayed because of dilution, the rumen having an estimated volume of 10 L/45 kg of body weight.26 However, the Cmax in this study was similar to that measured in horses given a comparable oral dose of Br.8 Ruminal epithelial cells express large conductance channels permeable to chloride27 and the rumen has a high chloride absorptive capacity, even against the net electrochemical gradient for the ion.28,29 It is likely that these high conductance channels are also responsible for overcoming a relatively lower initial Br concentration in the ruminal/gastric fluid compared with monogastric species. The t½ of Br in sheep, of approximately 14 days, is comparable with the 12 days in humans30 and the 12–24 days in the dog,31,32 but is considerably longer than the approximately 3 days in the horse.8 This long elimination phase is a function of slow clearance; Br is not metabolised and is subject to extensive renal tubular reabsorption, the net result being the anion is continually recycled throughout the body.31 In the classic PO pharmacokinetic profiles of monogastric species, secondary peaks are uncommon and are usually a function of unusual drug metabolism, in particular enterohepatic recycling. Bromide, however, is not metabolised, so possible causative mechanisms are limited to absorption, distribution and elimination. A study
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conducted in horses, using a similar PO dose of Br to that used in this study, produced a profile without secondary peaks in serum concentration.8 The secondary peaks phenomenon is possibly related to ongoing rumen and omasum redistribution, a function of bidirectional Br flux through chloride channels. Bromide has been shown in previous studies to move into ruminal fluid subsequent to IV administration.33 Similar secondary peaks were also seen following IV administration, albeit much less pronounced (Figure 1). However, it is notable that the IV peaks appeared to occur at similar time points to those seen following PO administration. Redistribution becomes even more complex when ruminant saliva volumes and the role of chloride in saliva production are taken into account. Ruminant saliva production is significantly higher than in most other species because it has an important role in providing fluid and buffer to the rumen. Saliva production in the sheep is estimated to vary between 3 and 10 L/day depending on feed type.34,35 One study estimated that 193 mL/h (4.6 L) of saliva is typically produced when eating dry forage, such as that consumed in this trial.36 This is approximately twice the estimated plasma volume for sheep. Transepithelial chloride movement is a driver of saliva production; however, the predominant chloride channels in salivary acinar cells favour Br secretion over chloride, with Br secreted in saliva at high concentrations.30,37–41 Given the large, but fluctuating volume of saliva production and the selective uptake of Br by salivary glands, it is possible that the fluctuations in serum Br concentration simply reflect the variation in saliva production at the time of sampling. The generalisation that Br is transported in the same way as chloride is useful, but probably overly simplistic. Just as Br is different electrochemically from chloride, there is also wide variation in ion selectivity, affinity/sensitivity and tissue distribution among anionic channels.42
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Further studies of the movement of Br in the rumen and saliva would be useful in better defining this process. Secondary peaks for other drugs have been identified in ruminants. Atypical peaks in plasma concentrations were seen at 8 and 13 h after PO indomethacin in sheep.43 The authors attributed these peaks to variable rates of absorption at multiple sites.43 However, gastrointestinal recirculation creating secondary peaks has been identified in monogastric species, including for meloxicam in humans.44,45 A series of peaks was also seen following PO tramadol in sheep, with those authors suggesting variable absorption, or delayed absorption from the drug binding to particulate matter, as the cause.46 The mean bioavailability of Br in this trial was 92%, which is much higher than the estimated bioavailability of 32–38% in the horse8 and 40% in the dog.31 This greater bioavailability in the sheep is probably related to the prolonged rumen residence time of ingesta.47 The Vd value of 0.286 ± 0.031 reflects the ECF space (although Vd data are not primary measures of physiological compartments, they do often correlate well) and approximates the Vd of 0.245 L/kg used as an estimate of ECF in the sheep.19 The calculated terminal volume of distribution (Vz), when equilibration with rumen fluid is assumed, was 0.393 ± 0.102 and 0.388 ± 0.037 L/kg for IV and PO administration, respectively. That these measures are similar is unsurprising because Vz is a proportionality factor related to concentrations during the log-linear phase of drug elimination, from which t½ is derived. Volume of distribution is the parameter used to calculate the LD, using the equation LD = V* Css, where Css is concentration steady state, or the effective concentration for a particular use (as determined by PD studies). The Vd value is most appropriate when Br is to be given as a PO bolus, and Vz in circumstances where it is to be given over a few days. Because of the long t½ following PO administration, the serum Br concentration fluctuated within a narrow range (∼200–400 mg/L) for 14 days. This long t½, with a two-fold difference between peak and trough values over a 2-week period confers great potential for therapeutic application, particularly prophylactic use. The study dose of Br (120 mg/kg) resulted in sustained Br concentrations approximating one-third of the lower boundary of the anticonvulsant range (1.0–2.0 mg/mL).48 Concentrations of Br that will prevent, attenuate or abolish PRGT tremor are unknown, although it would be assumed that in all but the most severe cases to be less than those required to protect against grand mal seizures. For PRGT and other neurological conditions causing tremor or seizures, it may be necessary to provide Br in a larger LD than was used in this study. The use of large oral LDs has been a problem in monogastric species and although it is anticipated that the large capacity of the rumen will mitigate these concerns, further research in this area is required before the extent of application of Br can be determined. However, even with the relatively large, acute PO dose given in this study there appeared to be no side effects and all animals continued eating after oral dosing. Concentrations of Br that will reduce anxiety and ameliorate the stress of transport are also unknown, but could rationally be assumed to be lower than those required to raise the ictal threshold. Some anxiolytic
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drugs have been associated with increased appetite and indeed assessment of the appetite of rodents in a novel situation can be used as an assessment of anxiolytic activity.9 Bromide has been shown to improve appetite in rats; such findings suggest Br may be well suited to preventing transport-induced inanition.10,11 It is well documented that an increase in NaCl intake will increase the excretion of Br. In dogs, the t½ of Br was 24 ± 7 d ays when they were fed a relatively low-salt diet and 15.2 days in beagles fed a high-salt diet.32,49 Although not quite a true antidote, common salt could be used to increase Br elimination; in circumstances where a shorter elimination time is required, the potential to hasten the elimination of a drug when it is no longer required, or desired, is important in food animals where residues are very much a concern. Because of the inherent problems of microbial biotransformation of drugs in the hostile ruminal fluid,50 the ovine liver’s ability to rapidly metabolise many sedative or anxiolytic drugs,51 and the often prohibitive cost of such drugs at the herd level, few realistic options have been available to producers in this important class of therapy. Though Br is by no means a new therapeutic in monogastric species, it is, however, a novel prospect for use in sheep. Several advantages of Br have now been indentified; Br is affordable, easy to administer, has good bioavailability and a long t½. It therefore has great potential for use in applications where a mild sedative, anxiolytic or anti-seizure drug is required.
References 1. Ryan M, Baumann RJ. Use and monitoring of bromides in epilepsy treatment. Pediatr Neurol 1999;21:523–528. 2. Thomas AB. Pharmacotherapy of mental illness: a historical analysis. Prog Neuropsychopharmacol Biol Psychiatry 2001;25:709–727. 3. Chang Y, Mellor DJ, Anderson TJ. Idiopathic epilepsy in dogs: owners’ perspectives on management with phenobarbitone and/or potassium bromide. J Small Anim Pract 2006;47:574–581. 4. Von Klopmann T, Rambeck B, Tipold A. Prospective study of zonisamide therapy for refractory idiopathic epilepsy in dogs. J Small Anim Pract 2007;48:134–138. 5. Trepanier LA, Van Schoick A, Schwark WS et al. Therapeutic serum drug concentrations in epileptic dogs treated with potassium bromide alone or in combination with other anticonvulsants: 122 cases (1992–1996). J Am Vet Med Assoc 1998;213:1449–1453. 6. Dowling PM. Management of canine epilepsy with phenobarbital and potassium bromide. Can Vet J 1994;35:724. 7. Ho ENM, Wan TSM, Wong ASY et al. Control of the misuse of bromide in horses. Drug Test Anal 2010;2:323–329. 8. Raidal SL, Edwards S. Pharmacokinetics of potassium bromide in adult horses. Aust Vet J 2008;86:187–193. 9. Britton DR, Britton KT. A sensitive open field measure of anxiolytic drug activity. Pharmacol Biochem Behav 1981;15:577–582. 10. Van Logten MJ, Wolthuis M, Rauws AG et al. Short-term toxicity study on sodium bromide in rats. Toxicology 1973;1:321–327. 11. Van Logten MJ, Wolthuis M, Rauws AG et al. Semichronic toxicity study of sodium bromide in rats. Toxicology 1974;2:257–267. 12. Dalziel JE, Finch SC, Dunlop J. The fungal neurotoxin lolitrem B inhibits the function of human large conductance calcium-activated potassium channels. Toxicol Lett 2005;155:421–426. 13. Imlach WL, Finch SC, Dunlop J et al. Structural determinants of lolitrems for inhibition of BK large conductance Ca2+-activated K+ channels. Eur J Pharmacol 2009;605:36–45. 14. Imlach WL, Finch SC, Dunlop J et al. The molecular mechanism of “ryegrass staggers”, a neurological disorder of K+ channels. J Pharmacol Exp Ther 2008;327:657–664.
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34. Carter RR, Allen OB, Grovum WL. The effect of feeding frequency and meal size on amounts of total and parotid saliva secreted by sheep. Br J Nutr 1990;63:305– 318. 35. Doyle P, Egan J, Thalen A. Parotid saliva of sheep. I: effects of level of intake and type of roughage. Aust J Agric Res 1982;33:573–584. 36. Osuji P, Gordon J, Webster A. Energy exchanges associated with eating and rumination in sheep given grass diets of different physical forms. Br J Nutr 1975;34:59–71. 37. Melvin JE. Chloride channels and salivary gland function. Crit Rev Oral Biol Med 1999;10:199–209. 38. Ullberg S, Appelgren LE, Clemedson CJ et al. A comparison of the distribution of some halide ions in the body. Biochem Pharmacol 1964;13:407–410, IN409IN412, 411–412. 39. Jentsch TJ, Stein V, Weinreich F et al. Molecular structure and physiological function of chloride channels. Physiol Rev 2002;82:503–568. 40. Melvin JE, Yule D, Shuttleworth T et al. Regulation of fluid and electrolyte secretion in the salivary gland acinar cells. Annu Rev Physiol 2005;67:445– 469. 41. Burgen ASV. Some processes concerned with the secretion of sodium, bromide and chloride in saliva. J Physiol 1963;169:663–678. 42. Alexander SP, Mathie A, Peters JA. Guide to receptors and channels (GRAC), 5th edition. Br J Pharmacol 2011;164(Suppl 1):S1–324. 43. Vinagre E, Encinas T, Ballesteros C, Rodríguez C. Pharmacokinetics of indomethacin after intraruminal administration to sheep. J Vet Med Series A 1999;46:361–366. 44. Türck D, Roth W, Busch U. A review of the clinical pharmacokinetics of meloxicam. Rheumatology 1996;35:13–16. 45. Burgos-Vargas R, Foeldvari I, Thon A et al. Pharmacokinetics of meloxicam in patients with juvenile rheumatoid arthritis. J Clin Pharmacol 2004;44:866–872. 46. De Sousa A, Santos A, Schramm S et al. Pharmacokinetics of tramadol and o-desmethyltramadol in goats after intravenous and oral administration. J Vet Pharmacol Ther 2008;31:45–51. 47. Cunningham F, Elliott J, Lees P. Comparative and veterinary pharmacology. Springer, Berlin, 2010. 48. Podell M, Fenner WR. Bromide therapy in refractory canine idiopathic epilepsy. J Vet Intern Med 1993;7:318–327. 49. March PA, Podell M, Sams RA. Pharmacokinetics and toxicity of bromide following high-dose oral potassium bromide administration in healthy Beagles. J Vet Pharmacol Ther 2002;25:425–432. 50. Baggot JD. The physiological basis of veterinary clinical pharmacology. WileyBlackwell, Oxford, 2008. 51. Boxenbaum H. Interspecies variation in liver weight, hepatic blood flow, and antipyrine intrinsic clearance: extrapolation of data to benzodiazepines and phenytoin. J Pharmacokinet Biopharmaceut 1980;8:165–176.
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15. Meierkord H, Grünig F, Gutschmidt U et al. Sodium bromide: effects on different patterns of epileptiform activity, extracellular pH changes and GABAergic inhibition. Naunyn-Schmiedeberg Arch Pharmacol 2000;361:25–32. 16. Goodwin JS, Katz RI, Kopin IJ. Effect of bromide on evoked release of monoamines from brain slices and intact atria. Nature 1969;221:556–557. 17. Suzuki S, Kawakami K, Nakamura F et al. Bromide, in the therapeutic concentration, enhances GABA-activated currents in cultured neurons of rat cerebral cortex. Epilepsy Res 1994;19:89–97. 18. Balcar VJ, Erdö SL, Joó F et al. Neurochemistry of GABAergic system in cerebral cortex chronically exposed to bromide in vivo. J Neurochem 1987;48:167–169. 19. Coghlan JA, Fan JSK, Scoggins BA et al. Measurement of extracellular fluid volume and blood volume in sheep. Aust J Biol Sci 1977;30:71–84. 20. Ward GM, Argenzio RH, Johnson JE. Evaluation of 82Br as an extracellular water tracer in ruminants. In: Isotope studies on the physiology of domestic animals. International Atomic Energy Agency, Vienna, 1972;73–80. 21. Reigner BG, Williams PE, Patel IH et al. An evaluation of the integration of pharmacokinetic and pharmacodynamic principles in clinical drug development: experience within Hoffmann La Roche. Clin Pharmacokinet 1997;33:142–152. 22. Lees P, Landoni MF, Giraudel J et al. Pharmacodynamics and pharmacokinetics of nonsteroidal anti-inflammatory drugs in species of veterinary interest. J Vet Pharmacol Ther 2004;27:479–490. 23. Toutain PL, Cester CC, Haak T et al. A pharmacokinetic/pharmacodynamic approach vs. a dose titration for the determination of a dosage regimen: the case of nimesulide, a Cox-2 selective nonsteroidal anti-inflammatory drug in the dog. J Vet Pharmacol Ther 2001;24:43–55. 24. Tietz NW. Fundamentals of clinical chemistry. WB Saunders, Philadelphia,1976. 25. Gibaldi M, Perrier, D. Pharmacokinetics. Marcel Dekker, New York, 1982. 26. Hinchcliff K, Jernigan A, Upson D et al. Ruminant pharmacology. Vet Clin North Am Food Anim Pract 1991;7:633. 27. Stumpff F, Martens H, Bilk S et al. Cultured ruminal epithelial cells express a large-conductance channel permeable to chloride, bicarbonate, and acetate. Pflugers Arch 2009;457:1003–1022. 28. Scott D. Absorption of chloride from the rumen of the sheep. Res Vet Sci 1970;11:291–293. 29. Dobson A, Phillipson AT. The absorption of chloride ions from the reticulorumen sac. J Physiol London 1958;140:94–104. 30. Harden RMG, Alexander W, Shimmins J et al. A comparison between the gastric and salivary concentration of iodide, pertechnetate, and bromide in man. Gut 1969;10:928. 31. Trepanier LA, Babish JG. Pharmacokinetic properties of bromide in dogs after the intravenous and oral administration of single doses. Res Vet Sci 1995;58:248– 251. 32. Trepanier LA, Babish JG. Effect of dietary chloride content on the elimination of bromide by dogs. Res Vet Sci 1995;58:252–255. 33. Coghlan JP, Fan JS, Scoggins BA et al. Measurement of extracellular fluid volume and blood volume in sheep. Aust J Biol Sci 1977;30:71–84.
(Accepted for publication 12 June 2013)
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Pharmacokinetics of bromide in adult sheep following oral and intravenous administration TA Quast, MD Combs and SH Edwards*
Objective To determine the pharmacokinetics of bromide in sheep after single intravenous (IV) and oral (PO) doses. Procedure Sixteen Merino sheep were randomly assigned to two treatment groups and given 120 mg/kg bromide, as sodium bromide IV or potassium bromide PO. Serum bromide concentrations were determined by colorimetric spectrophotometry. Results After IV administration the maximum concentration (Cmax) was 822.11 ± 93.61 mg/L, volume of distribution (Vd) was 0.286 ± 0.031 L/kg and the clearance (Cl) was 0.836 ± 0.255 mL/h/ kg. After PO administration the Cmax was 453.86 ± 43.37 mg/L and the time of maximum concentration (Tmax) was 108 ± 125 h. The terminal half-life (t½) of bromide after IV and PO administration was 387.93 ± 115.35 h and 346.72 ± 94.05 h, respectively. The oral bioavailability (F) of bromide was 92%. No adverse reactions were noted in either treatment group during this study. The concentration versus time profiles exhibited secondary peaks, suggestive of gastrointestinal cyclic redistribution of the drug. Conclusions and clinical relevance When administered PO, bromide in sheep has a long half-life (t½) of approximately 14 days, with good bioavailability. Potassium bromide is a readily available, affordable salt with a long history of medical use as an anxiolytic, sedative and antiseizure therapy in other species. There are a number of husbandry activities and flock level neurological conditions, including perennial ryegrass toxicosis, in which bromide may have therapeutic or prophylactic application. Keywords
bromide; pharmacokinetics; sheep
Abbreviations AUC0-∞, area under the curve; AUMC0-∞, area under the first moment curve; Cmax, maximum concentration; λz, terminal elimination rate constant; ECF, extracellular fluid; LD, loading dose; PK, pharmacokinetics; PD, pharmacodynamics; PRGT, perennial ryegrass toxicosis; t½, terminal elimination half-life; Tmax, time to maximum concentration; Vd, initial volume of distribution; Vz, terminal volume of distribution. Aust Vet J 2015;93:20–25
doi: 10.1111/avj.12285
B
romide has been a therapeutic option for human epilepsy since its efficacy in the management of seizures was first discovered in the middle of the 19th century.1 In humans it has also been used for a wide variety of other applications, including as a sedative and an anxiolytic.2
*Corresponding author. School of Animal and Veterinary Science, Charles Sturt University, Wagga Wagga, New South Wales, Australia; [email protected]
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In veterinary medicine, potassium bromide (KBr) has found renewed favour in recent years as a therapeutic agent. In Australia, KBr is registered in dogs for the treatment of idiopathic epilepsy and it is being increasingly used worldwide for seizure therapy as it is associated with fewer side-effects than phenobarbitone.3–6 It is also used extensively off-label by horse owners as a calmative for nervous horses; however, to date little research has been undertaken as to its efficacy and appropriate application.7,8 The potential uses of Br in sheep have not been previously investigated. Because of its tranquillising and anxiolytic actions, Br has the potential to address animal welfare and production issues associated with a range of husbandry procedures such as road and sea transportation, anxiety associated with shearing and inanition associated with stress and introduction of new feeds.9–11 Bromide also has a potential use as a therapeutic agent for neurological diseases that affect sheep at the herd level. For example, the perennial ryegrass toxin, lolitrem B, was recently identified as a calciumactivated potassium channels antagonist, depolarising the neuronal membrane and thereby increasing neuronal excitability.12–14 Bromide’s action via chloride channels results in membrane hyperpolarisation and Br also appears to potentiate GABAergic inhibition.1,6,15–18 These results suggest Br could be used prophylactically to reduce the incidence of perennial ryegrass toxicosis (PRGT) associated with lolitrem B. The distribution of Br in sheep has been partially elucidated; IV 82Br was used to estimate ovine extracellular fluid (ECF) volume and was found to exhibit delayed distribution kinetics.19,20 Essentially, Br is initially confined within the vascular space, with extravascular movement into interstitial fluid being slow, taking 2–3 h to equilibrate with the ECF. Its equilibration with rumen fluid is even slower, with only 4–5% entering the rumen at 3 h and taking 24 h for full equilibration to be reached. In the course of drug development, both pharmacokinetic (PK) and pharmacodynamic (PD) studies are conducted in order to guide determination of rational dosage schedules (dose and administration interval) for subsequent clinical trials. From PK studies in a small number of animals, using a test dose (not necessarily a predicted therapeutic dose), the concentration versus time profile is generated. From these simple plots, estimates of the necessary PK parameters may be derived, which are subsequently used to determine a dose regimen. Target drug concentrations (within the therapeutic range) are determined from PD studies, involving dose–response experiments (in vitro and in vivo dose titration). The use of such a PK–PD approach offers rational guidance in attempting to streamline the drug development process, with the aim of reducing animal wastage stemming from large-scale dose-titration studies.21–23 Moreover, once the
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Of prime importance in this study was to determine the PK parameters of most clinical relevance. A relatively long terminal elimination half-life (t½) is important for an extended duration of action; t½ is also used to estimate withholding periods, vitally important with any drug used in food animals. Volume of distribution (Vd) is the parameter used to calculate the loading dose (LD), a large dose administered to reach target drug concentrations quickly. For the first time, this study investigated the pharmacokinetics of Br in sheep, as the basis for future studies of its therapeutic applications. Materials and methods Animals Sixteen 6-year-old merino ewes (weight 49.5–67 kg; average body condition score > 2) were allocated equally to IV and PO treatment groups by simple systematic randomisation (all sheep were mixed in a pen then put through a race, with every second sheep assigned to pen B. Allocation of treatment to pen A and B was decided by a coin toss). Animals were placed in individual feeding pens and fed twice daily on a ration of oats and lupins, with ad libitum hay and water. Estimated chloride content of the oats and lupins was 0.11% and 0.4%, respectively. Indwelling 16G, 32-cm IV cannulas (Braun, Certo Splittocan 335, Melsungen, Germany) were placed in the left jugular vein and secured with a 2/0 polypropylene suture. A 25-cm low-volume IV extension set (BMDi TUTA, NSW, Aust) was connected to the catheter hub and the area was then bandaged. Sodium bromide (NaBr; Sigma-Aldrich, St Louis, MO, USA) and potassium bromide (KBr; Sigma-Aldrich) solutions were prepared using sterile water. The prepared NaBr solution was then filtered through a microfilter (0.22 μ, MILLEX GP, Cork, Ireland). Sodium and potassium salts were administered to dose the sheep with 120 mg/kg of Br (154.6 mg/kg NaBr or 178.8 mg/kg KBr). All serum concentrations are for Br. KBr is the most readily available form of Br for oral therapy and NaBr was used for the IV study because of cardiotoxicity associated with potassium. Both salts were fully disassociated in solution, so PK differences were not expected. Administration route Intravenous. The NaBr solution was administered through the cephalic vein using a 21G needle over a period of 1 min. Sheep were restrained in a ‘seated’ position. Blood samples were collected at 0, 1, 5, 10, 15, 20 and 30 min and then at 1, 2, 3, 4, 6, 8, 10, 12 and 24 h. Samples thereafter were collected at 12 h intervals to 240 h and then at 24 h intervals to 336 h. A final sample was taken at 528 h. For each sample, the initial 2 mL of blood collected was discarded and a sterile syringe used to withdraw 5 mL of blood, which was then placed into a plain separator blood tube (Vacuette, Greiner Bio-one, Kremsmünster, Austria). The cannula was flushed with 3 mL of 5% heparinised saline after each collection. Each blood sample was left to stand for 30 min
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before centrifugation at 2000g for 5 min. Serum was harvested and stored at −20°C until analysis. PRODUCTION ANIMALS
PK parameters are known, it is a simple matter to use PK–PD integration to generate a putative dose regimen for multiple applications of a particular drug. Should a therapeutic have a novel application potential, the new target concentration is applied to the existing PKs.
Oral. The KBr solution was administered via an orogastric tube, which was then flushed with 500 mL water. Blood samples were collected at 0, 1, 2, 3, 4, 6, 8, 10, 12 and 24 h, then at 12 h intervals to 240 h and then at 24 h intervals to 336 h. A final sample was taken at 504 h. When collecting blood samples at 1 h through to 10 h the rumen was auscultated over the caudodorsal blind sack to determine if the high salt load had affected rumen motility. Determination of serum bromide concentrations Serum Br concentrations were determined by colorimetric spectrophotometry as previously described,24 with some modification. Briefly, 0.35 mL of serum was added to 3.15 mL of 10% trichloroacetic acid (Sigma-Aldrich) in a 10-mL centrifuge tube, vortexed, then centrifuged for 15 min at 2000g. Next, 2.5 mL of supernatant was mixed with 0.25 mL of 0.5% Au2Cl6 (Sigma-Aldrich) and left to stand for 30 min. Absorbance was measured with a spectrophotometer at 440 nm. The standard curve was linear in the range of 25–5000 μg/ mL, R2 = 0.9992. The lower limit of quantification was 25 μg/mL. Pharmacokinetics Maximum concentration (Cmax) of Br and time to Cmax (Tmax) were determined directly from the data. Other PK parameters were determined for each sheep by non-compartmental analysis using a commercial software program (Topfit 2.0, Gustav Fischer Verlag). Area under the curve (AUC0-∞) and area under the first moment curve (AUMC0-∞) were calculated by the linear trapezoidal rule;25 the terminal elimination rate constant (λz) was calculated by means of loglinear regression. Clearance (Cl) was calculated using the equation:
Cl =
Dose AUC0−∞
Mean residence time (MRT) was calculated using the equation:
MRT =
AUMC0−∞ AUC0−∞
The initial volume of distribution (Vd) was calculated using the equation below, with C0 estimated by extrapolation of the drug disposition curve from 2 h (assumed equilibration with ECF) to time zero.
Vd =
Dose C0
The terminal volume of distribution (Vz) at pseudo-equilibrium was calculated using the equation:
Vz =
Cl λz
The t½ was calculated using the equation:
t1/2 =
0.69315 λz
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PRODUCTION ANIMALS Whereas parameters Cmax, Tmax, and AUC (where bioavailability is not absolute) are expected to differ when given IV or PO, t½ should be the same, regardless of the route of administration. A t-test of the hypothesis of no difference between the t½ population means was performed. All results are expressed as mean ± standard deviation. Results Animals No discernable neurological effects were seen in sheep in the PO group. No alteration in rumen motility was auscultated and animals continued to eat and drink. Assessment of any acute neurological effects correlating with peak Br concentration following IV administration was difficult because the sheep were held in the ‘seated’ position
throughout the initial 20 min for ease of sampling. Following IV injection, all sheep walked back to their individual pens and observers subjectively reported a mild tranquilising effect for approximately 1–2 h post-injection. Pharmacokinetics The relevant non-compartmental pharmacokinetic parameters derived from this study are summarised in Table 1. The concentration– time profiles for IV and PO serum Br are shown in Figures 1 and 2, respectively. The t½ of Br in sheep following PO administration was 14.4 days and the IV t½ was 16.2 days; however, the difference between groups was not statistically significant (T = 0.7832, df = 14, P = 0.4466).
Table 1. Pharmacokinetic parameters (mean ± SD) of bromide after intravenous and oral administration to eight sheep at a dose of 120 mg/kg
Pharmacokinetic variable
Intravenous administration (mean ± SD)
Oral administration (mean ± SD)
822.11 ± 93.61 − 157221.8 ± 52681.53 545.5 ± 226.1 0.836 ± 0.255 0.286 ± 0.031 0.393 ± 0.102 387.93 ± 115.35 −
453.86 ± 43.37 108 ± 124.86 143948.9 ± 26156.16 413.4 ± 150 − − 0.388 ± 0.037 346.72 ± 94.05 0.92
Cmax (mg/L) Tmax (h) AUC0-∞ (mg*h/L) MRT0-∞ (h) Cl (mL/h/kg) Vd (L/kg) Vz (L/kg) t½ (h) F
AUC0-∞, area under the curve; Cl, clearance; Cmax, maximum concentration; F, oral bioavailability; MRT0-∞, mean residence time; t½, terminal elimination half-life; SD, standard deviation; Tmax, time of maximum concentration; Vd, initial volume of distribution, Vz, terminal volume of distribution at pseudo-equilibrium.
Figure 1. Serum concentration (mean ± SD) of bromide after intravenous administration to eight sheep at a dose of 120 mg/kg.
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Figure 2. Serum concentration (mean ± SD) of bromide after oral administration to eight sheep at a dose of 120 mg/kg. Note multiple peaks occurring after 24 h.
There were numerous pronounced peaks in the PO concentration versus time curve following the initial absorption phase. These peaks approached the Cmax seen on day 1, but occurred many days later (Figure 2). Indeed, some sheep had Tmax values well beyond the initial PO absorption phase. Similar but smaller peaks were also seen with IV Br (Figure 1). Discussion Bromide was absorbed rapidly following PO administration. Prior to this study, it was predicted that absorption might be delayed because of dilution, the rumen having an estimated volume of 10 L/45 kg of body weight.26 However, the Cmax in this study was similar to that measured in horses given a comparable oral dose of Br.8 Ruminal epithelial cells express large conductance channels permeable to chloride27 and the rumen has a high chloride absorptive capacity, even against the net electrochemical gradient for the ion.28,29 It is likely that these high conductance channels are also responsible for overcoming a relatively lower initial Br concentration in the ruminal/gastric fluid compared with monogastric species. The t½ of Br in sheep, of approximately 14 days, is comparable with the 12 days in humans30 and the 12–24 days in the dog,31,32 but is considerably longer than the approximately 3 days in the horse.8 This long elimination phase is a function of slow clearance; Br is not metabolised and is subject to extensive renal tubular reabsorption, the net result being the anion is continually recycled throughout the body.31 In the classic PO pharmacokinetic profiles of monogastric species, secondary peaks are uncommon and are usually a function of unusual drug metabolism, in particular enterohepatic recycling. Bromide, however, is not metabolised, so possible causative mechanisms are limited to absorption, distribution and elimination. A study
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conducted in horses, using a similar PO dose of Br to that used in this study, produced a profile without secondary peaks in serum concentration.8 The secondary peaks phenomenon is possibly related to ongoing rumen and omasum redistribution, a function of bidirectional Br flux through chloride channels. Bromide has been shown in previous studies to move into ruminal fluid subsequent to IV administration.33 Similar secondary peaks were also seen following IV administration, albeit much less pronounced (Figure 1). However, it is notable that the IV peaks appeared to occur at similar time points to those seen following PO administration. Redistribution becomes even more complex when ruminant saliva volumes and the role of chloride in saliva production are taken into account. Ruminant saliva production is significantly higher than in most other species because it has an important role in providing fluid and buffer to the rumen. Saliva production in the sheep is estimated to vary between 3 and 10 L/day depending on feed type.34,35 One study estimated that 193 mL/h (4.6 L) of saliva is typically produced when eating dry forage, such as that consumed in this trial.36 This is approximately twice the estimated plasma volume for sheep. Transepithelial chloride movement is a driver of saliva production; however, the predominant chloride channels in salivary acinar cells favour Br secretion over chloride, with Br secreted in saliva at high concentrations.30,37–41 Given the large, but fluctuating volume of saliva production and the selective uptake of Br by salivary glands, it is possible that the fluctuations in serum Br concentration simply reflect the variation in saliva production at the time of sampling. The generalisation that Br is transported in the same way as chloride is useful, but probably overly simplistic. Just as Br is different electrochemically from chloride, there is also wide variation in ion selectivity, affinity/sensitivity and tissue distribution among anionic channels.42
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Further studies of the movement of Br in the rumen and saliva would be useful in better defining this process. Secondary peaks for other drugs have been identified in ruminants. Atypical peaks in plasma concentrations were seen at 8 and 13 h after PO indomethacin in sheep.43 The authors attributed these peaks to variable rates of absorption at multiple sites.43 However, gastrointestinal recirculation creating secondary peaks has been identified in monogastric species, including for meloxicam in humans.44,45 A series of peaks was also seen following PO tramadol in sheep, with those authors suggesting variable absorption, or delayed absorption from the drug binding to particulate matter, as the cause.46 The mean bioavailability of Br in this trial was 92%, which is much higher than the estimated bioavailability of 32–38% in the horse8 and 40% in the dog.31 This greater bioavailability in the sheep is probably related to the prolonged rumen residence time of ingesta.47 The Vd value of 0.286 ± 0.031 reflects the ECF space (although Vd data are not primary measures of physiological compartments, they do often correlate well) and approximates the Vd of 0.245 L/kg used as an estimate of ECF in the sheep.19 The calculated terminal volume of distribution (Vz), when equilibration with rumen fluid is assumed, was 0.393 ± 0.102 and 0.388 ± 0.037 L/kg for IV and PO administration, respectively. That these measures are similar is unsurprising because Vz is a proportionality factor related to concentrations during the log-linear phase of drug elimination, from which t½ is derived. Volume of distribution is the parameter used to calculate the LD, using the equation LD = V* Css, where Css is concentration steady state, or the effective concentration for a particular use (as determined by PD studies). The Vd value is most appropriate when Br is to be given as a PO bolus, and Vz in circumstances where it is to be given over a few days. Because of the long t½ following PO administration, the serum Br concentration fluctuated within a narrow range (∼200–400 mg/L) for 14 days. This long t½, with a two-fold difference between peak and trough values over a 2-week period confers great potential for therapeutic application, particularly prophylactic use. The study dose of Br (120 mg/kg) resulted in sustained Br concentrations approximating one-third of the lower boundary of the anticonvulsant range (1.0–2.0 mg/mL).48 Concentrations of Br that will prevent, attenuate or abolish PRGT tremor are unknown, although it would be assumed that in all but the most severe cases to be less than those required to protect against grand mal seizures. For PRGT and other neurological conditions causing tremor or seizures, it may be necessary to provide Br in a larger LD than was used in this study. The use of large oral LDs has been a problem in monogastric species and although it is anticipated that the large capacity of the rumen will mitigate these concerns, further research in this area is required before the extent of application of Br can be determined. However, even with the relatively large, acute PO dose given in this study there appeared to be no side effects and all animals continued eating after oral dosing. Concentrations of Br that will reduce anxiety and ameliorate the stress of transport are also unknown, but could rationally be assumed to be lower than those required to raise the ictal threshold. Some anxiolytic
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drugs have been associated with increased appetite and indeed assessment of the appetite of rodents in a novel situation can be used as an assessment of anxiolytic activity.9 Bromide has been shown to improve appetite in rats; such findings suggest Br may be well suited to preventing transport-induced inanition.10,11 It is well documented that an increase in NaCl intake will increase the excretion of Br. In dogs, the t½ of Br was 24 ± 7 d ays when they were fed a relatively low-salt diet and 15.2 days in beagles fed a high-salt diet.32,49 Although not quite a true antidote, common salt could be used to increase Br elimination; in circumstances where a shorter elimination time is required, the potential to hasten the elimination of a drug when it is no longer required, or desired, is important in food animals where residues are very much a concern. Because of the inherent problems of microbial biotransformation of drugs in the hostile ruminal fluid,50 the ovine liver’s ability to rapidly metabolise many sedative or anxiolytic drugs,51 and the often prohibitive cost of such drugs at the herd level, few realistic options have been available to producers in this important class of therapy. Though Br is by no means a new therapeutic in monogastric species, it is, however, a novel prospect for use in sheep. Several advantages of Br have now been indentified; Br is affordable, easy to administer, has good bioavailability and a long t½. It therefore has great potential for use in applications where a mild sedative, anxiolytic or anti-seizure drug is required.
References 1. Ryan M, Baumann RJ. Use and monitoring of bromides in epilepsy treatment. Pediatr Neurol 1999;21:523–528. 2. Thomas AB. Pharmacotherapy of mental illness: a historical analysis. Prog Neuropsychopharmacol Biol Psychiatry 2001;25:709–727. 3. Chang Y, Mellor DJ, Anderson TJ. Idiopathic epilepsy in dogs: owners’ perspectives on management with phenobarbitone and/or potassium bromide. J Small Anim Pract 2006;47:574–581. 4. Von Klopmann T, Rambeck B, Tipold A. Prospective study of zonisamide therapy for refractory idiopathic epilepsy in dogs. J Small Anim Pract 2007;48:134–138. 5. Trepanier LA, Van Schoick A, Schwark WS et al. Therapeutic serum drug concentrations in epileptic dogs treated with potassium bromide alone or in combination with other anticonvulsants: 122 cases (1992–1996). J Am Vet Med Assoc 1998;213:1449–1453. 6. Dowling PM. Management of canine epilepsy with phenobarbital and potassium bromide. Can Vet J 1994;35:724. 7. Ho ENM, Wan TSM, Wong ASY et al. Control of the misuse of bromide in horses. Drug Test Anal 2010;2:323–329. 8. Raidal SL, Edwards S. Pharmacokinetics of potassium bromide in adult horses. Aust Vet J 2008;86:187–193. 9. Britton DR, Britton KT. A sensitive open field measure of anxiolytic drug activity. Pharmacol Biochem Behav 1981;15:577–582. 10. Van Logten MJ, Wolthuis M, Rauws AG et al. Short-term toxicity study on sodium bromide in rats. Toxicology 1973;1:321–327. 11. Van Logten MJ, Wolthuis M, Rauws AG et al. Semichronic toxicity study of sodium bromide in rats. Toxicology 1974;2:257–267. 12. Dalziel JE, Finch SC, Dunlop J. The fungal neurotoxin lolitrem B inhibits the function of human large conductance calcium-activated potassium channels. Toxicol Lett 2005;155:421–426. 13. Imlach WL, Finch SC, Dunlop J et al. Structural determinants of lolitrems for inhibition of BK large conductance Ca2+-activated K+ channels. Eur J Pharmacol 2009;605:36–45. 14. Imlach WL, Finch SC, Dunlop J et al. The molecular mechanism of “ryegrass staggers”, a neurological disorder of K+ channels. J Pharmacol Exp Ther 2008;327:657–664.
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34. Carter RR, Allen OB, Grovum WL. The effect of feeding frequency and meal size on amounts of total and parotid saliva secreted by sheep. Br J Nutr 1990;63:305– 318. 35. Doyle P, Egan J, Thalen A. Parotid saliva of sheep. I: effects of level of intake and type of roughage. Aust J Agric Res 1982;33:573–584. 36. Osuji P, Gordon J, Webster A. Energy exchanges associated with eating and rumination in sheep given grass diets of different physical forms. Br J Nutr 1975;34:59–71. 37. Melvin JE. Chloride channels and salivary gland function. Crit Rev Oral Biol Med 1999;10:199–209. 38. Ullberg S, Appelgren LE, Clemedson CJ et al. A comparison of the distribution of some halide ions in the body. Biochem Pharmacol 1964;13:407–410, IN409IN412, 411–412. 39. Jentsch TJ, Stein V, Weinreich F et al. Molecular structure and physiological function of chloride channels. Physiol Rev 2002;82:503–568. 40. Melvin JE, Yule D, Shuttleworth T et al. Regulation of fluid and electrolyte secretion in the salivary gland acinar cells. Annu Rev Physiol 2005;67:445– 469. 41. Burgen ASV. Some processes concerned with the secretion of sodium, bromide and chloride in saliva. J Physiol 1963;169:663–678. 42. Alexander SP, Mathie A, Peters JA. Guide to receptors and channels (GRAC), 5th edition. Br J Pharmacol 2011;164(Suppl 1):S1–324. 43. Vinagre E, Encinas T, Ballesteros C, Rodríguez C. Pharmacokinetics of indomethacin after intraruminal administration to sheep. J Vet Med Series A 1999;46:361–366. 44. Türck D, Roth W, Busch U. A review of the clinical pharmacokinetics of meloxicam. Rheumatology 1996;35:13–16. 45. Burgos-Vargas R, Foeldvari I, Thon A et al. Pharmacokinetics of meloxicam in patients with juvenile rheumatoid arthritis. J Clin Pharmacol 2004;44:866–872. 46. De Sousa A, Santos A, Schramm S et al. Pharmacokinetics of tramadol and o-desmethyltramadol in goats after intravenous and oral administration. J Vet Pharmacol Ther 2008;31:45–51. 47. Cunningham F, Elliott J, Lees P. Comparative and veterinary pharmacology. Springer, Berlin, 2010. 48. Podell M, Fenner WR. Bromide therapy in refractory canine idiopathic epilepsy. J Vet Intern Med 1993;7:318–327. 49. March PA, Podell M, Sams RA. Pharmacokinetics and toxicity of bromide following high-dose oral potassium bromide administration in healthy Beagles. J Vet Pharmacol Ther 2002;25:425–432. 50. Baggot JD. The physiological basis of veterinary clinical pharmacology. WileyBlackwell, Oxford, 2008. 51. Boxenbaum H. Interspecies variation in liver weight, hepatic blood flow, and antipyrine intrinsic clearance: extrapolation of data to benzodiazepines and phenytoin. J Pharmacokinet Biopharmaceut 1980;8:165–176.
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15. Meierkord H, Grünig F, Gutschmidt U et al. Sodium bromide: effects on different patterns of epileptiform activity, extracellular pH changes and GABAergic inhibition. Naunyn-Schmiedeberg Arch Pharmacol 2000;361:25–32. 16. Goodwin JS, Katz RI, Kopin IJ. Effect of bromide on evoked release of monoamines from brain slices and intact atria. Nature 1969;221:556–557. 17. Suzuki S, Kawakami K, Nakamura F et al. Bromide, in the therapeutic concentration, enhances GABA-activated currents in cultured neurons of rat cerebral cortex. Epilepsy Res 1994;19:89–97. 18. Balcar VJ, Erdö SL, Joó F et al. Neurochemistry of GABAergic system in cerebral cortex chronically exposed to bromide in vivo. J Neurochem 1987;48:167–169. 19. Coghlan JA, Fan JSK, Scoggins BA et al. Measurement of extracellular fluid volume and blood volume in sheep. Aust J Biol Sci 1977;30:71–84. 20. Ward GM, Argenzio RH, Johnson JE. Evaluation of 82Br as an extracellular water tracer in ruminants. In: Isotope studies on the physiology of domestic animals. International Atomic Energy Agency, Vienna, 1972;73–80. 21. Reigner BG, Williams PE, Patel IH et al. An evaluation of the integration of pharmacokinetic and pharmacodynamic principles in clinical drug development: experience within Hoffmann La Roche. Clin Pharmacokinet 1997;33:142–152. 22. Lees P, Landoni MF, Giraudel J et al. Pharmacodynamics and pharmacokinetics of nonsteroidal anti-inflammatory drugs in species of veterinary interest. J Vet Pharmacol Ther 2004;27:479–490. 23. Toutain PL, Cester CC, Haak T et al. A pharmacokinetic/pharmacodynamic approach vs. a dose titration for the determination of a dosage regimen: the case of nimesulide, a Cox-2 selective nonsteroidal anti-inflammatory drug in the dog. J Vet Pharmacol Ther 2001;24:43–55. 24. Tietz NW. Fundamentals of clinical chemistry. WB Saunders, Philadelphia,1976. 25. Gibaldi M, Perrier, D. Pharmacokinetics. Marcel Dekker, New York, 1982. 26. Hinchcliff K, Jernigan A, Upson D et al. Ruminant pharmacology. Vet Clin North Am Food Anim Pract 1991;7:633. 27. Stumpff F, Martens H, Bilk S et al. Cultured ruminal epithelial cells express a large-conductance channel permeable to chloride, bicarbonate, and acetate. Pflugers Arch 2009;457:1003–1022. 28. Scott D. Absorption of chloride from the rumen of the sheep. Res Vet Sci 1970;11:291–293. 29. Dobson A, Phillipson AT. The absorption of chloride ions from the reticulorumen sac. J Physiol London 1958;140:94–104. 30. Harden RMG, Alexander W, Shimmins J et al. A comparison between the gastric and salivary concentration of iodide, pertechnetate, and bromide in man. Gut 1969;10:928. 31. Trepanier LA, Babish JG. Pharmacokinetic properties of bromide in dogs after the intravenous and oral administration of single doses. Res Vet Sci 1995;58:248– 251. 32. Trepanier LA, Babish JG. Effect of dietary chloride content on the elimination of bromide by dogs. Res Vet Sci 1995;58:252–255. 33. Coghlan JP, Fan JS, Scoggins BA et al. Measurement of extracellular fluid volume and blood volume in sheep. Aust J Biol Sci 1977;30:71–84.
(Accepted for publication 12 June 2013)
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